This application focuses on two Solute Carrier Proteins 1A5 and 38A2 (SLC1A5/38A2) membrane proteins that affects cellular metabolism through their role in glutamine uptake and amino acid influx. We identified SLC1A5/38A2 as associated proteins with the ubiquitin ligase RNF5. Treatment of breast cancer (BCa) cells with select chemotherapeutic drugs induces an association between RNF5 and SLC1A5 that results in ubiquitination-dependent degradation of SLC1A5/38A2. As a direct consequence of their loss, BCa exhibits decreased levels of glutamine uptake and reduced mTOR, 4EBP1, and S6K phosphorylation, which inhibits colony formation and increases cell death. Our preliminary data using a tumor microarray (TMA) of more than 500 BCa samples demonstrate that SLC1A5/38A2 expression is low in estrogen receptor-positive and node- negative tumors, thereby associated with good prognosis. Notably, in about 25% of samples, high RNF5 expression is associated with low SLC1A5/38A2 expression, raising the possibility that SLC1A5/38A2 may be regulated by RNF5 in these BCa tumors. These observations suggest that SLC1A5/38A2 may play an important role in the development and the response of BCa to select therapies, in part through RNF5- dependent mechanisms. Our proposed studies aim at understanding the regulatory mechanisms underlying the regulation and function of SLC1A5/38A2 in BCa, and the significance of these proteins for BCa development and response to therapy. Specifically we will: (1) Identify RNF5-dependent and -independent transcriptional, translational, and post-translational events regulating SLC1A5/38A2 availability and activity in subpopulations of BCa cells. (2) Establish the biological significance of SLC1A5/38A2 expression in BCa cells for cellular metabolism, mitochondrial dynamics and function, autophagy, growth, and response to therapy. (3) Determine the relationship between BCa expression of SLC1A5/38A2 and RNF5, the response to treatment, and disease outcome. Our proposed studies will establish the importance of glutamine metabolism in our understanding of BCa biology, offer novel approaches for the stratification of BCa to therapy while providing the foundation for the development of novel therapeutic modalities.